Article

Effect of thyme and oregano essential oils on the shelf

life of salmon and seaweed burgers

D Dolea1 , A Rizo2, A Fuentes1, JM Barat1 and

I Fernández-Segovia1

Abstract
The effect of different essential oils on the quality and shelf life of fish and seaweed burgers during storage
was evaluated. For this purpose, thyme and oregano essential oils were selected at a concentration of 0.05%
(v/w). Three types of salmon and seaweed burgers were prepared: without essential oil, burgers with red
thyme essential oil (0.05% (v/w)) and burgers with oregano essential oil (0.05% (v/w)), which were vacuum
packaged and stored at 4  C for 17 days. Physicochemical and microbiological analyses were carried out
periodically throughout storage. The addition of both essential oils did not have any effect on the evolution of
the pH, the moisture content or texture parameters. Only the thyme essential oil managed to slightly slow
down the increase of total volatile basic nitrogen and trimethylamine nitrogen. The samples with oregano
essential oil and especially those with thyme essential oil showed minor oxidation. The salmon and seaweed
burgers without essential oils and those which contained oregano essential oil showed a faster increase of
mesophilic counts than those which had thyme essential oil, but no noticeable improvement was observed in
the shelf life of the burgers with thyme essential oil. To improve the shelf life of the fish and seaweed burgers,
it would be necessary to increase the concentration of both essential oils.

Keywords
Algae, antimicrobial agents, antioxidants, essential oils, fish and fish products
Date received: 1 September 2017; accepted: 22 January 2018

for their antioxidant capacity (Farvin and Jacobsen,

INTRODUCTION
In the last decades, an increase in the use of seaweeds as
food ingredients in western countries has been
observed. This fact is closely linked to the excellent
nutritional composition and functional properties that
seaweeds exhibit. They are rich in proteins, essential
amino acids and minerals (Peinado et al., 2014;
Rupérez, 2002). These marine plants have low fat con-
tent which implies the low caloric value that they add.
It is important to highlight the significant amount of
fibre which seaweeds provide (Cofrades et al., 2008;
Gómez-Ordóñez et al., 2010). The phenolic compounds
present in the seaweeds are also noticeable, responsible
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2013). This property is important for human health,
because the oxidative processes are involved in a wide
variety of diseases. In addition, the lipid oxidation
plays an important role in the fatty food spoilage,
where the use of synthetic additives is very often neces-
sary. Seaweeds could be an alternative to the use of this
type of additives, constituting a natural source of
antioxidants.
The bioactive compounds present in seaweeds have
also been proven to exhibit antimicrobial action against
1
Departamento de Tecnologı́a de Alimentos, Universitat
Politècnica de València, Valencia, Spain
2
Instituto de Agroquı́mica y Tecnologı́a de Alimentos (IATA-CSIC),
Valencia, Spain
Corresponding author:
I Fernández-Segovia, Departamento de Tecnologı́a de Alimentos,
Universitat Politècnica de València, Camino de Vera s/n, Valencia
46022, Spain.
Email: isferse1@tal.upv.es
Food Science and Technology International 0(0)
gram-positive and gram-negative bacteria (Gupta and
Abu-Ghannam, 2011). This property would allow sea-
weeds to replace chemical preservatives.
At present the incorporation of seaweeds as a func-
tional ingredient constitutes an interesting alternative in
the development of new healthy food products, which
are both natural and have high nutritional value.
In the search for alternatives to the synthetic preser-
vatives or antioxidant, the essential oils (EOs) obtained
from plants have also been widely studied. The anti-
microbial and antioxidant activity of EOs is due to
their high levels of phenolic compounds. In this sense,
thyme is characterized by high concentrations of
thymol and p-cymene, and to a lesser extent a-pinene
and linalool. Oregano has high contents of carvacrol
and p-cymene, with other compounds, such as
thymol, in lower concentrations. Clove is characterized
by its high content in eugenol, while cinnamon is rich in
cinnamic aldehyde, among others. Rosemary shows
important contents of borneol, camphor, a-pinene
and 1,8-cineol (Ballester-Costa et al., 2013; Perricone
et al., 2015; Pesavento et al., 2015). The effectivity in
the inhibition of microbial growth and the antioxidant
capacity of some EOs, such as rosemary, oregano,
thyme or cinnamon, on fish and meat products have
been proven in several studies (Abdollahzadeh et al.,
2014; Kostaki et al., 2009; Kykkidou et al., 2009;
Mexis et al., 2009; Pesavento et al., 2015).
The aim of this work was to evaluate the effect of
different EOs on the quality and shelf life of salmon and
seaweed burgers during storage.
MATERIALS AND METHODS
Materials
Fish. Aquacultured salmon (Salmo salar) of commercial
size (weighing 2–3 kg) from a Norwegian farm
(Hallvard Leroy, Bergen, Norway) was used as raw
material. The fish were purchased in a local supermar-
ket in Valencia (Spain). The specimens were headed,
gutted and filleted, and two fillets were obtained from
each fish. On arrival at the laboratory, the fillets were
frozen at 20  C until processing and analysis, for a
total period of 72 h.
Seaweed. Dehydrated and powdered seaweeds were
used as raw material. Four types of seaweeds (kombu
(Laminaria ochroleuca), wakame (Undaria pinnatı´fida),
espagueti de mar (Himanthalia elongata) and agar-agar
(Gelidium sesquipedale)) were provided by the company
Porto-Muiños (Cerceda, A Coruña, Spain).
EOs. Three EOs were studied: red thyme EO
(Thymus zygis L.), cinnamon bark EO (Cinnamomum
zeylanicum) and oregano EO (Origanum vulgare L.).
They were provided by the company Ernesto Ventós,
S.A. (Sant Just Desvern, Barcelona, Spain). According
2
to the manufacturer’s specifications, red thyme EO was
obtained from the entire flowering plant (without root),
the cinnamon bark EO was obtained from the dried
bark and had a content of cinnamic aldehyde >60%
and safrole <0.1%, and the oregano EO was obtained
from the flowering tops and the phenol contents ranged
from 60 to 75%.
Selection of EO concentrations
A preliminary sensory study was conducted to select
the concentration of each EO which was sensorially
acceptable. The concentrations of red thyme, cinnamon
bark and oregano EOs are shown in Table 1. To select
the concentration of each EO to be added to the bur-
gers for the final study, seven trained panellists were
used (two men and five women), members of the
Food Technology Department at the Universitat
Politècnica de València, which had participated in pre-
vious studies of development of burgers, pâtes and
sausages made of seaweeds and fish.
Burger samples were presented to the panel in two
different sessions. In the first one, nine samples were
evaluated (the three EOs at 0.5, 0.25 or 0.10% v/w).
In the second one, six samples were tested (the three
EOs at 0.05 or 0.025% v/w) (Table 1). The panellists
had to decide by consensus if each of the samples eval-
uated was sensorially acceptable or not. Based on the
results obtained in this preliminary study, as will be
explained in the ‘Results and Discussion’ section, two
EOs were selected (red thyme and oregano) at the con-
centration of 0.050 ml/100 g sample. Sample prepara-
tion was conducted following the procedure described
below.
Sample preparation
Salmon fillets were thawed during 24 h at 4  C. Salmon
skin and remaining bones were removed and it was cut
into pieces of 3 cm  3 cm approximately. The portions
were randomly divided into three batches. Batch 1 was
used to prepare control samples (Ct samples), which
consisted of burgers made of salmon and seaweeds
Table 1. Essential oil (EO) concentrations used in the
preliminary study
EO Concentration (ml EO/100 g sample)
Red thyme 0.50 0.25 0.10 0.050 0.025
Cinnamon bark 0.50 0.25 0.10 0.050 0.025
Oregano 0.50 0.25 0.10 0.050 0.025
First session Second session
 Dolea et al.

without EOs. Portions in batches 2 and 3 were used to
prepare salmon–seaweeds burgers with red thyme EO
(0.050 ml/100 g sample) (T samples) and with oregano
EO (Og samples) (0.050 ml/100 g sample), respectively
(Figure 1).
Portions of each batch were weighed and minced in
the ThermomixÕ (Vorwerk & Co., Wuppertal,
Germany) for 45 s. Then, a mixture of the four sea-
weeds in powder were added in the necessary amount
up to a final content of 1% kombu, 1% wakame, 3%
sea spaghetti and 1% agar-agar (w/w), and the fish with
the seaweeds were homogenized for 45 s in the
ThermomixÕ , until obtaining an homogeneous paste
(sample Ct). This methodology was based on previous
studies carried out by the authors (data not published).
Burgers with EOs were prepared in the same way as
control samples, incorporating the EO at the same time
as the seaweed mixture (Figure 1).
Finally, the paste obtained was divided into 30 g por-
tions, which were introduced into a mould to give the
typical shape of small burgers. All samples were
vacuum packaged (two burgers/bag) using high barrier
bags and stored at 4  C for 17 days.
Analytical determinations
Physicochemical and microbiological analyses of the
burgers were performed on days 0, 3, 7, 10, 14 and 17
of refrigerated storage. Three bags of each batch were
randomly taken on each sampling day (n ¼ 3). The ana-
lyses were performed in duplicate on each sample.
Physicochemical analyses
Moisture contents were determined according to the
AOAC method 950.46 (1997).
A pH meter (Crison Instruments, S.A., Barcelona,
Spain) was used to measure pH values, previously
homogenizing the samples in distilled water
(weight:volume ratio 1:10).
The total volatile basic nitrogen (TVB-N) and tri-
methylamine nitrogen (TMA-N) contents were deter-
mined by steam distillation according to the method
described by Malle and Tao (1987). Thirty grams of
sample was weighed and homogenized with 60 ml of
trichloroacetic acid solution 7.5% (w/v), centrifuged
at 3000 rpm/min for 5 min. The supernatant was filtered
through a Whatman No. 1 filter, obtaining the extract

Thawed salmon fillets (Salmo salar)

Cut into portinons

Batch 2

Mincing 45 s Mincing 45 s Mincing 45 s

Seaweed mixture Seaweed mixture Seaweed mixture

+ thyme EO + oregano EO

Homogenisation 45 s Homogenisation 45 s Homogenisation 45 s

Burger shaping Burger shaping Burger shaping

Vacuum packaging and Vacuum packaging and Vacuum packaging and

storage at 4ºC for 17 days storage at 4ºC for 17 days storage at 4ºC for 17 days

Samples Ct Samples T Samples Og

Figure 1. Flow diagram of sample preparation.

3
Food Science and Technology International 0(0)
of the sample. For TVB-N determination, 5 ml of
NaOH 10% (w/v) was added to 25 ml of the extract
and it was submitted to steam distillation in a Unit
Pro Nitro M, SELECTA (Barcelona, Spain). The
ammonia was collected in 15 ml of boric acid at 4%
(w/v) and titrated with sulphuric acid 0.01 N. Results
were expressed as mg N/100 g sample. The same pro-
cedure was followed for TMA-N analysis, except for
the addition of 20 ml of formaldehyde solution 35%
(v/v) to the extract before distillation, in order to
block primary and secondary amines.
The thiobarbituric acid (TBA) index was measured
using a spectrophotometric method with some minor
modifications (Tarladgis et al., 1960) to evaluate oxida-
tion stability during chilled storage. The method is
based on the measurement of absorbance at 530 nm
of a chromogen formed by reaction between thiobarbi-
turic acid and malondialdehyde (MDA) (one of the
main compounds formed as a result of lipid oxidation).
Texture measurements
Texture profile analysis (TPA) was performed by use of a
Texture Analyser TA.XT2Õ (Stable Micro Systems,
Surrey, UK), equipped with a load cell of 250 N.
Samples were obtained by cutting out pieces of 2 cm  2 cm
from the central part of each burger. For TPA analysis a
flat-ended cylindrical plunger (7.5 cm diameter) was used.
The plunger was pressed into the sample at a constant
speed of 1 mm/s until it reached 50% of the sample
height, in order to simulate jaw movement during masti-
cation (Bourne, 1978). Force–distance curves were pro-
cessed in order to obtain the texture parameters:
hardness, cohesiveness, elasticity, chewiness and resilience.
Microbiological analyses
Mesophilic bacteria and Enterobacteriaceae were
determined according to the methods given by the
ISO standards 4833:2003 and 21528-2:2004, respect-
ively. All the analyses were performed in duplicate
and the results were expressed as log cfu/g. All the cul-
ture media were provided by Scharlau Chemie, S.A.
(Barcelona, Spain).
Statistical analysis
During the storage study, the data relating to each par-
ameter were analysed with a multifactor ANOVA to
evaluate the effect of the EO and of the storage time
and their interactions. All the physicochemical and
microbiological parameters were considered as depend-
ent variables and type of EO and the storage time were
the factors in these analyses. Data are reported as
mean  standard deviation. Statistical treatment of the
4
data was performed using the Statgraphics Centurion
software (Statpoint Technologies, Inc., Warrenton,
VA, USA).
RESULTS AND DISCUSSION
Selection of EO concentrations
The EOs have strong odours and flavours, which is why it
is necessary to check that the amount incorporated to a
food product is acceptable from a sensorial point of view.
In the first session of the sensorial analysis, panellists
evaluated the three EOs (red thyme, cinnamon bark
and oregano) at the highest concentrations (0.5, 0.25
and 0.1 ml EO/100 g sample) in the burgers, as mentioned
above. They concluded that for those concentrations, the
burgers were not acceptable for any of the EOs.
In the second session, the concentrations tested were
0.05 and 0.025 ml EO/100 g sample, for the three EOs.
Panellists established that the cinnamon EO conferred a
strong, odd flavour to the burgers, not being acceptable
at any of the concentrations used. For the red thyme
and oregano EOs for both concentrations, they con-
cluded that, although the burgers had typical flavours
of these EOs, they could be acceptable for consumers.
Based on these results, the use of cinnamon EO was
rejected for the final study. For the red thyme and ore-
gano EOs, the concentration of 0.05 ml/100 g was chosen,
since it was the highest amount at which the burgers
obtained may be sensorially acceptable. Nevertheless,
this study should be completed with a further sensory
analysis to check the consumer’s acceptation with hedo-
nic trials using a high number of non-trained panellists.
It is important to note that in other studies carried
out on swordfish with thyme EO, contents of 0.1%
(v/w) in fresh fish were sensorially acceptable.
However, in other works on squid rings, concentrations
equal to or over 0.075% of thyme and oregano EOs in
the coating mixture, gave a strong odour in the final
product, using concentrations equal to or below 0.05%
(Sanjuás-Rei et al., 2012), which agrees with this work.
Quality and shelf life study
Changes in the physicochemical parameters during
storage. Values of pH and moisture content in the
three types of burgers (Ct, T and Og) during cold storage
are shown in Table 2. Initially, pH was approximately 6.2,
with no significant differences between the three types of
samples. This parameter slightly decreased from day 0 to
day 14, showing a slight increase from this sampling day
onwards, as has been observed in other fish studies (Cai
et al., 2015; Mexis et al., 2009).
The initial decrease could be due to the growth of
lactic acid bacteria with the subsequent lactic acid for-
mation. The final increase could be due to basic
 Dolea et al.

Table 2. Evolution of pH, moisture and texture parameters in salmon and seaweed burgers: without
essential oils (Ct), with thyme essential oil (T) and with oregano essential oil (Og), during 17 days of
storage at 4  C. (means and standard deviations)

Days of
Parameters storage Ct T Og

pH 0 6.19  0.01 6.21  0.01 6.20  0.01

3 6.14  0.03 6.24  0.03 6.22  0.04
7 6.10  0.00 6.14  0.04 6.11  0.01
10 6.08  0.01 6.03  0.01 6.01  0.01
14 6.02  0.01 6.03  0.05 5.99  0.03
17 6.05  0.03 6.06  0.00 6.05  0.02
Moisture 0 63.2  0.4 63.2  0.9 62.9  0.3
3 62.6  0.4 63.5  0.8 62.5  0.2
7 63.1  0.8 63.4  0.3 62.9  0.2
10 64.1  0.5 65.4  0.7 64.2  0.1
14 65.1  0.6 65.3  0.1 64.5  0.1
17 64.1  0.4 63.9  0.2 63.4  0.1
Texture
Hardness (N) 0 3.27  0.01 3.91  0.01 3.68  0.01
3 7.08  0.07 4.79  0.08 4.8  0.8
7 5.1  0.8 4.2  0.1 52
10 3.76  0.06 5.6  0.4 5.2  0.3
14 52 4.7  0.7 62
17 4.5  0.7 5.1  0.4 62
Adhesiveness (g s) 0 0.09  0.00 0.12  0.01 0.07  0.01
3 0.29  0.02 0.19  0.03 0.16  0.06
7 0.13  0.03 0.13  0.03 0.16  0.09
10 0.11  0.01 0.26  0.04 0.18  0.05
14 0.2  0.1 0.15  0.01 0.19  0.06
17 0.19  0.09 0.29  0.09 0.17  0.08
Elasticity 0 0.54  0.00 0.60  0.01 0.55  0.01
3 0.70  0.03 0.64  0.04 0.63  0.02
7 0.65  0.03 0.65  0.03 0.5  0.2
10 0.66  0.03 0.78  0.08 0.65  0.09
14 0.76  0.07 0.70  0.01 0.6  0.2
17 1.4  0.1 1.40  0.09 1.4  0.6
Chewiness (N) 0 0.89  0.00 1.23  0.01 0.99  0.05
3 2.6  0.1 1.6  0.1 1.6  0.4
7 1.9  0.2 1.6  0.1 1.6  0.9
10 1.5  0.3 2.58  0.02 1.96  0.01
14 2.8  0.7 1.9  0.3 2.3  0.9
17 3.44  0.04 4.1  0.7 54
Cohesiveness 0 0.50  0.00 0.52  0.00 0.49  0.01
3 0.53  0.01 0.54  0.01 0.54  0.01
7 0.54  0.04 0.56  0.02 0.62  0.09
10 0.54  0.01 0.59  0.01 0.57  0.04
14 0.57  0.03 0.59  0.01 0.6  0.1
17 0.6  0.2 0.60  0.02 0.59  0.02
(continued)

5
Food Science and Technology International 0(0)

Table 2. Continued

Days of
Parameters storage Ct T Og

Resilience 0 0.15  0.00 0.15  0.00 0.15  0.00

3 0.15  0.00 0.15  0.00 0.15  0.00
7 0.15  0.01 0.17  0.01 0.24  0.08
10 0.16  0.00 0.15  0.00 0.18  0.01
14 0.16  0.00 0.18  0.00 0.21  0.07
17 11 0.18  0.02 0.18  0.02

compounds formation as a consequence of fish spoil-

(a)
age. The EOs incorporation did not affect the pH 70
Control
values throughout the storage, with no significant dif- 60 Thyme

mg N / 100 g sample
Oregano
ferences between samples. These results agree with 50
those observed by Kykkidou et al. (2009), where the 40
thyme EO incorporated to fresh fish did not have any 30
effect on the pH evolution. 20
Moisture values varied during storage with no clear 10
trend. T samples exhibited values slightly higher than 0
the other two types of burgers, although the statistical 0 5 10 15 20
Storage time (days)
analysis showed no statistical differences between sam-
(b)
ples (p > 0.05). 35
Control
mg N/100 g sample

The TVB-N, TMA-N and TBA index evolution are 30 Thyme

Oregano
shown in Figure 2(a) to (c), respectively. The TVB-N is 25
widely used as a fish spoilage index, since an increase in 20
this parameter has been associated with the spoilage 15
specific bacterial growth and with endogenous enzyme 10
activity, which entail the loss of quality in fish (Özyurt 5
et al., 2009). The acceptability limit of TVB-N varies 0
depending on the fish species, season, region, age or 0 5 10 15 20
Storage time (days)
sex. This parameter also depends on whether the fish (c)
is fresh or processed. 1
mg malonaldehyde/kg sample

Control
Thyme
According to Regulation 2074/2005, the European 0.8 Oregano
Commission (2005) considers an acceptable value of
0.6
35 mg N/100 g for S. salar. In control sample (Ct), the
value of this parameter increased from 18.9 up to 32 mg 0.4
N/100 g at day 10 of storage. The acceptability limit
0.2
was exceeded on day 14, showing an important increase
through to day 17 of study, where values were signifi- 0
0 5 10 15 20
cantly higher than those with EOs. Burgers with ore-
Storage time (days)
gano EO showed a similar pattern to the Ct samples. In
samples with thyme EO, the TVB-N levels kept under
the acceptability limit until day 17, although at day 14, Figure 2. Evolution of TVB-N (a), TMA-N (b) and TBA
T samples exhibited values very close to Ct and Og index (c), in salmon and seaweed burgers: without EOs
(control), with thyme EO (thyme) and with oregano EO
samples. From these results, it can be concluded that
(oregano), during 17 days of storage at 4  C. (means and
the EOs only showed a slight effect at the end of the standard deviations). Dashed line shows the acceptability
studied period, but they would not be able to inhibit or limit.
delay spoilage, from the point of view of this param-
eter, since values close to the acceptability limits were
obtained practically at the same time for the three types during the storage time can be observed for all the sam-
of samples. ples. Samples with EOs showed lower values of this
Initial values of TMA-N range from 3 to 4 mg parameter than Ct samples, as occurred with TVB-N.
N/100 g of sample. In Figure 2(b) a progressive increase Og samples exhibited a similar pattern to the control

6
 Dolea et al.

samples, except for the last day of analysis, where the
values were lower in burgers with oregano EO. T bur-
gers had a slower evolution during the first days of
storage. However, from day 10 these samples reached
similar values to Ct and Og burgers.
Regarding the TBA index, the initial values were low
for the three types of burgers, remaining stable until
day 7 of analysis. From this day onwards, a gradual
increase was observed in all samples. The Ct samples
exhibited a significantly higher increase than those with
thyme and oregano EOs. This fact shows the antioxi-
dant effect of the EOs, which agrees with other studies
on thyme and oregano (Kostaki et al., 2009; Kykkidou
et al., 2009; Mexis et al., 2009). Marino et al. (1999)
reported the important antioxidant potential of thyme
extract due to the high concentrations of phenolic com-
pounds, including carvacrol, thymol, p-cymene, among
others. Different acceptability limits have been given
for this parameter in different fish species. No absolute
limit for TBA index has been established for fish or fish
products in general. According to Conell (1995), the
limit would be 1–2 mg MDA/kg; whereas other authors
have given higher values. Although Ct samples showed
TBA values significantly higher than those of burgers
with EOs, it is important to note that the value reached
by control samples at the end of storage was 0.599 mg
MDA/kg. Taking into account the lowest acceptability
limits for this parameter (around 1 mg MDA/kg), that
value is considered to be low. It can be concluded that
any of the burgers evaluated exhibited an important
oxidation degree. This demonstrates that, in addition
observed on the evolution of fish hardness (Cai et al.,
2015). In this work, since the fish was previously
minced, the texture evolution has not reflected the
spoilage of the product and, therefore, although the
EOs would have had a preservative action, this could
not be reflected on the texture study.
Changes in the microbial parameters during
storage. Evolution of mesophilic and
Enterobacteriaceae counts in the three types of burgers
(Ct, T and Og) during cold storage is shown in
Figure 3(a) and (b), respectively. Levels of mesophilic
counts of 7 log cfu/g have been established as the
acceptability limit, based on the criteria established by
the International Commission on Microbiological
Specifications for Foods (1986) and numerous studies
of fish shelf life (Cai et al., 2015; Kykkidou et al., 2009;
Özyurt et al., 2012). At the beginning of the study,
the mesophilic counts in the three types of samples
were around 5 log cfu/g. A progressive increase of the
growth of this microorganism was observed until day
10 for all samples, with no significant differences
between them. However, from day 10, Ct and Og bur-
gers exhibited a faster increase compared with T
(a)
10
Control
8
Thyme
Oregano
log cfu/g sample

to the antioxidant effect of the EOs, the vacuum packa- 6

ging would play a decisive role in burger preservation 4

against oxidation. It is also important to mention that
in numerous studies, the antioxidant capacity of sea- 2

weeds has been proven (Farvin and Jacobsen 2013; 0

López-López et al., 2009). 0 5 10 15 20
Storage time (days)
The textural parameters are shown in Table 2.
The initial values of hardness were similar in the three (b)
6
Control
burgers. For the three types of samples, a fluctuation of Thyme
this parameter could be observed throughout the stor- Oregano
log cfu/g sample

4
age, without a clear trend. Therefore, the EOs incorp-
oration had no effect on this textural parameter. This
behaviour could be due to the fact that the preparation 2
of the burgers included mincing the fish; consequently,
the muscle structure was destroyed from day 0.
Adhesiveness values also varied throughout the time 0
0 5 10 15 20
period with no trend. Elasticity and chewiness values Storage time (days)
remained constant until day 14 of storage, slightly
increasing at the end of the study. Cohesiveness Figure 3. Evolution of mesophilic bacteria (a) and
and resilience remained stable during all the time. Enterobacteriaceae (b) in salmon and seaweed burgers:
These results indicate that the EOs did not affect the without EOs (control), with thyme EO (thyme) and with
evaluated texture parameters or their evolution through oregano EO (oregano), during 17 days of storage at 4  C
the storage time. In work carried out on red drum fillets (means and standard deviations). Dashed line shows the
with different types of EOs, a positive effect was acceptability limit.

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Food Science and Technology International 0(0)

samples. At the end of the study, all samples reached Table 3. F-ratio values and significance levels obtained in
levels near the acceptability limit (107 cfu/g). the multifactor ANOVA for the physicochemical and
The oregano EO, therefore, at the concentration microbiological parameters according to factors: type of
used in this study, did not exert any inhibitory effect sample (S), storage time (t) and their interaction (S  t)
on mesophilic growth. Although the thyme EO slows S T St
down the microbial growth, there was no noticeable
ns
improvement of the T burger’s shelf life. In order to pH 1.87 62.57*** 2.42ns
delay microbial spoilage, a higher concentration of Moisture 6.63** 19.73*** 0.79ns
both EOs would be necessary. However, the increase TVB-N 0.32ns 15.78*** 1.64ns
of the EOs concentration would affect the sensory TMA-N 4.50* 50.03*** 1.67ns
quality. TBA index 1535.78*** 5871.80*** 283.60***
Based on the results of mesophilic growth, the shelf Texture
life of the burgers (Ct, Og or T) would be lower than or Hardness 0.23ns 2.98* 1.47ns
equal to 17 days. Adhesiveness 0.89ns 3.79* 1.68ns
Regarding the Enterobacteriaceae, fluctuations of
Elasticity 0.46ns 24.09*** 0.23ns
counts were observed during storage with no clear
Cohesiveness 0.27ns 3.45* 0.58ns
trend. Although C samples showed slightly higher
counts on some sampling days, at the end of the Chewiness 0.02ns 7.21*** 0.65ns
study, Enterobacteriaceae levels were similar in the Resilience 0.81ns 1.05ns 1.00ns
three types of burgers, which demonstrated that Mesophilic bacteria 14.95*** 470.79*** 16.78***
thyme and oregano EOs did not have antimicrobial Enterobacteriaceae 3.34* 16.48*** 2.13*
effect under the conditions used in this study. ns: non-significant; TBA: thiobarbituric acid; TMA-N: trimethyla-
In work carried out on trout fillets using oregano EO mine nitrogen; TVB-N: total volatile basic nitrogen.
at 0.4% (v/w), an important reduction in the mesophilic *p < 0.05; **p < 0.01; ***p < 0.001.
and Enterobacteriaceae counts was observed, extending
the shelf life by 3–4 days (Mexis et al., 2009). Similar F-ratio and the significance level of factors and their
results were obtained in a study conducted on swordfish interactions. The F-ratio represents the quotient
with thyme (0.1%), with a shelf life extension of about between variability due to the effect considered and
three days. the residual variance. A higher value of F-ratio means
The results of the present study demonstrated that a more marked effect of that factor (type of sample or
the oregano or thyme EOs added to salmon and sea- storage time) on a variable (parameters analysed).
weed burgers at 0.05% (v/w) achieved a reduction in The statistical analysis showed that the use of thyme
the degree of sample oxidation, but they did not slow or oregano EOs significantly affected the TBA index
down microbial growth to the necessary levels to extend and mesophilic counts. Storage time had a significant
the burger’s shelf life. effect in practically all the analysed variables, although,
Given that product shelf life was limited by the TVB- as has been mentioned before, some parameters varied
N contents and mesophilic growth, both EOs should be throughout the storage time with no clear trend.
incorporated at higher concentrations to be effective in Regarding interaction between the factors, it is import-
the improvement of hygienic quality of burger made ant to highlight that it was significant for the TBA
from salmon and seaweed. Taking into account that, index and mesophilic counts, which demonstrated
from a sensorial point of view, this increase would not that the evolution of both parameters was different
be possible, combinations of the different EOs and/or depending on the type of sample.
combinations with other preservation methods would
be interesting, since several authors have demonstrated
CONCLUSIONS
that they can have a synergetic effect on food preserva-
tion (Calo et al., 2015; Cherrat et al., 2014; De Oliveira Cinnamon EO confers an intense odour and flavour,
et al., 2015). In this way, low concentrations of EOs even at low concentrations (0.025 v/w), which make it
could be used to achieve microbial inhibition without unsuitable for use as a natural preservative in salmon
compromising burger sensory quality. and seaweed burgers. Red thyme and oregano EOs
could be incorporated to these burgers at concentra-
tions of 0.05% (v/w) conferring a typical odour and
Multifactor ANOVA
flavour, but which may be sensorially acceptable.
An ANOVA multifactor was conducted to study the Oregano EO at 0.05% (v/w) in the burgers does not
influence of the EOs and the storage time, on the par- affect the changes of TVB-N or TMA-N during cold
ameters analysed in the burgers. Table 3 shows the storage. The red thyme EO at the same concentration

8

slows down the increase of both parameters; neverthe-
less, this decrease in the TVB-N content is not enough
to significantly extend burger’s shelf life.
Moisture content, pH and texture of the product are
not affected by the red thyme or oregano EOs. Both
EOs have a marked influence on the inhibition of
burger oxidation. The antioxidant capacity of seaweed
together with the vacuum packaging, also protect bur-
gers against oxidation, meaning that oxidation is not a
spoilage process in this product.
Enterobacteriaceae growth is not affected by the
presence of both EOs at the concentrations studied,
while the addition of red thyme EO causes a decrease
in mesophilic growth. However, this inhibition is not
enough to increase burger’s shelf life.
In order to extend the product shelf life, both EOs
had to be used at higher concentrations, but this is not
possible from a sensorial point of view. The combin-
ation of the different EOs and/or combinations with
other preservation techniques would be interesting,
since they may have a synergetic effect on burger pres-
ervation. In this way, low concentrations of EOs could
be used to achieve microbial inhibition without com-
promising the burger sensory quality.
DECLARATION OF CONFLICTING INTERESTS
The author(s) declared no potential conflicts of interest with
respect to the research, authorship, and/or publication of this
article.
FUNDING
The author(s) disclosed receipt of the following financial sup-
port for the research, authorship, and/or publication of this
article: The experiment reported here is a part of a project
financially supported by Universitat Politècnica de València
(‘SaPesAl’ (UPV-FE-2014-55)), which the authors gratefully
acknowledge. The translation of this paper was funded by the
Universitat Politècnica de València, Spain.
ORCID ID
D Dolea http://orcid.org/0000-0001-8278-7967
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